Methods for neural conversion of human embryonic stem cells

Abstract

The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. Specifically, methods are provided for obtaining neural tissue, floor plate cells, and placode including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons, motor neurons, and sensory neurons. Further, neural plate tissue obtained using methods of the present inventions are contemplated for use in co-cultures with other tissues as inducers for shifting differentiation pathways, i.e. patterning.

What is claimed is: 
     
       1. A composition comprising a cell population, two inhibitors of Small Mothers Against Decapentaplegic (SMAD) protein signaling, and an activator of Sonic Hedgehog (SHH) signaling, wherein the cell population comprises in vitro differentiated cells, wherein at least about 10% of the differentiated cells are neuronal floor plate cells, wherein the two inhibitors of SMAD protein signaling are selected from the group consisting of: a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542), and derivatives of SB431542. 
     
     
       2. The composition of  claim 1 , wherein said neuronal floor plate cells express at least one marker selected from the group consisting of SHH, FOXA2, Netrin-1, F-Spondin, and combinations thereof. 
     
     
       3. The composition of  claim 1 , wherein the two inhibitors of SMAD protein signaling comprise an inhibitor of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforming growth factor beta (TGFβ)/Activin-Nodal signaling. 
     
     
       4. The composition of  claim 3 , wherein the inhibitor of BMP signaling is selected from the group consisting of a disulfide-linked homodimer of Noggin, Dorsomorphin, and LDN-193189. 
     
     
       5. The composition of  claim 3 , wherein the inhibitor of BMP signaling comprises LDN-193189. 
     
     
       6. The composition of  claim 3 , wherein the inhibitor of TGFβ/Activin-Nodal signaling comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542). 
     
     
       7. The composition of  claim 1 , wherein the activator of SHH signaling comprises a SHH protein. 
     
     
       8. The composition of  claim 7 , wherein the SHH protein comprises a recombinant protein, wherein the amino acid sequence of the recombinant protein comprises the amino acid sequence of mouse Sonic Hedgehog N-terminal fragment. 
     
     
       9. The composition of  claim 7 , wherein the SHH protein is selected from the group consisting of Sonic hedgehog (SHH) and Sonic C25II.